Several cellular pathways involved in RNA-mediated gene suppression have been described, each distinguished by a characteristic pathway and specific components. Generally, RNA-mediated gene suppression involves a double-stranded RNA (dsRNA) intermediate that is formed intramolecularly within a single RNA molecule or intermolecularly between two RNA molecules. This longer dsRNA intermediate is processed by a ribonuclease of the RNase III family (Dicer or Dicer-like ribonuclease) to one or more small double-stranded RNAs, one strand of which is incorporated by the ribonuclease into the RNA-induced silencing complex (“RISC”). Which strand is incorporated into RISC is believed to depend on certain thermodynamic properties of the double-stranded small RNA, such as those described by Schwarz et al. (2003) Cell, 115:199-208, and Khvorova et al. (2003) Cell, 115:209-216.
The siRNA pathway involves the non-phased cleavage of a longer double-stranded RNA intermediate to small interfering RNAs (“siRNAs”). The size of siRNAs is believed to range from about 19 to about 25 base pairs, but common classes of siRNAs include those containing 21 base pairs or 24 base pairs. See, for example, Hamilton et al. (2002) EMBO J., 21:4671-4679.
The microRNA pathway involves microRNAs (“miRNAs”), non-protein coding RNAs generally of between about 19 to about 25 nucleotides (commonly about 20-24 nucleotides in plants) that guide cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways; see Ambros et al. (2003)RNA, 9:277-279. Naturally occurring miRNAs are derived from a primary transcript (“pri-miRNA”) that is naturally processed to a shorter transcript (“pre-miRNA”) which itself is further processed to the mature miRNA. For a recent review of miRNA biogenesis in both plants and animals, see Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385. Gene regulation of biological pathways by miRNAs can occur at multiple levels and in different ways, including regulation of single or multiple genes, regulation of transcriptional regulators, and regulation of alternative splicing; see Makeyev & Maniatis (2008) Science, 319:1789-1790. Various utilities of miRNAs, their precursors, their recognition sites, and their promoters are described in detail in co-assigned U.S. Patent Application Publication 2006/0200878 A1, specifically incorporated by reference herein, which include: (1) the expression of a native miRNA or miRNA precursor sequence to suppress a target gene; (2) the expression of an engineered (non-native) miRNA or miRNA precursor sequence to suppress a target gene; (3) expression of a transgene with a miRNA recognition site, wherein the transgene is suppressed when the corresponding mature miRNA is expressed, either endogenously or transgenically; and (4) expression of a transgene driven by a miRNA promoter.
In the trans-acting siRNA (“ta-siRNA”) pathway, miRNAs serve to guide in-phase processing of siRNA primary transcripts in a process that requires an RNA-dependent RNA polymerase for production of a double-stranded RNA precursor; trans-acting siRNAs are defined by lack of secondary structure, a miRNA target site that initiates production of double-stranded RNA, requirements of DCL4 and an RNA-dependent RNA polymerase (RDR6), and production of multiple perfectly phased ˜21-nt small RNAs with perfectly matched duplexes with 2-nucleotide 3′ overhangs (see Allen et al. (2005) Cell, 121:207-221; Vazquez et al. (2004) Mol. Cell, 16:69-79).
The phased small RNA (“phased sRNA”) pathway (see PCT patent application PCT/US2007/019283, published as WO 2008/027592) is based on an endogenous locus termed a “phased small RNA locus”, which transcribes to an RNA transcript forming a single foldback structure that is cleaved in phase in vivo into multiple small double-stranded RNAs (termed “phased small RNAs”) capable of suppressing a target gene. In contrast to siRNAs, a phased small RNA transcript is cleaved in phase. In contrast to miRNAs, a phased small RNA transcript is cleaved by DCL4 or a DCL4-like orthologous ribonuclease (not DCL1) to multiple abundant small RNAs capable of silencing a target gene. In contrast to the ta-siRNA pathway, the phased small RNA locus transcribes to an RNA transcript that forms hybridized RNA independently of an RNA-dependent RNA polymerase and without a miRNA target site that initiates production of double-stranded RNA.
Gene suppression mediated by small RNAs processed from natural antisense transcripts has been reported in at least two pathways. In the natural antisense transcript small interfering RNA (“nat-siRNA”) pathway (Borsani et al. (2005) Cell, 123:1279-1291), siRNAs are generated by DCL1 cleavage of a double-stranded RNA formed between the antisense transcripts of a pair of genes (cis-antisense gene pairs). A similar natural anti-sense transcript microRNA (“nat-miRNA”) pathway (Lu et al. (2008) Proc. Natl. Acad. Sci. USA, 105: 4951-4956) has also been reported. In metazoan animals, small RNAs termed Piwi-interacting RNAs (“piRNAs”) have been reported to also have gene-silencing activity (Lau et al. (2006) Science, 313:363-367; O'Donnell & Boeke (2007) Cell, 129:37-44).